Genomic DNA has to be replicated before every cycle of cell division. Although replicative DNA polymerases have a built-in proofreading mechanism to minimize errors during replication, occasionally mismatch due to replication-error occurs. Mismatch repair systems to prevent mutations from replicative errors exist in most organisms. E. coli has a methyl-directed mismatch repair system comprising MutS, MutL and MutH proteins. Homologues of MutS and MutL proteins are also found in humans. Mutations in MutS or MutL homologs have been identified in 90% of the hereditary nonpolyposis colorectal cancers. By Oct. 2001, our group determined the crystal structures of mismatch repair proteins MutH, a conserved 40KD ATPase fragment of MutL and its complexes with nucleotides, the ATPase domain of a human MutL homologue, PMS2, alone and complexed with ATP and nonhydrolyzeable ATP analog, and the 190 Kd Taq MutS alone, complexed with DNA, and as a ternary complex with DNA and ADP/Mg2+. Last year (2001-2002), (1) based on the crystal structures, we constructed 47 MutS, MutL and MutH E. coli mutants, carried out in vitro and in vivo (in collaboration with J. Miller at UCLA) biochemical studies, and found that every mutant that fails to repair mismatch is deficient in preventing homeologous recombination. Our studies also revealed that the non-specific DNA binding property of MutL is required for mismatch repair after the DNA incision by MutH and that the structural domains in MutS work cooperatively to achieve maximal differential binding of heteroduplex versus homoduplex rather than maximal DNA binding. (2) The MutL ATPase active site shares conserved sequence motifs with DNA topoisomerases, Hsp90 and bacterial and mitochondrial kinases. Many of these proteins are potential drug targets. We found that despite the similar ATP binding pocket among these enzymes and a shared requirment of Mg ion, each of them differs in monovalent ion preference in the ATP binding pocket. Based on structure and sequence comparison between MutL and a rat mitochondrial protein kinase, we made a point mutation in MutL and converted MutL from using any monomalent ion to Na+ specifically. We have also determined the crystal structures of the wildtype and mutant MutL proteins and observed a Na+ for K+ exchange. We propose that the monovalent ion specificity may be exploited for future drug design. We have also determined the crystal structure of a hemimethylated GATC binding proten, E. coli SeqA, bound to the DNA. This project was derived from our research on how MutH recognizes hemimethylated GATC sequence and targets mismatch repair to the daughter strand specifically. The structure of SeqA-DNA complex reveals that the recognition of the methylated adenine is achieved by protein mainchain atoms via close van der Waals contacts. Most interestingly, the structure suggests a mechanism for SeqA to sequestrate DNA replication origin, oriC, from being used prematurely, repeatedly or asynchronously. A new family of DNA polymerases, the Y-family, has recently been identified. They differ from the previously known DNA polymerases in the primary sequence and in the ability of lesion-bypass and error-prone DNA synthesis. In collaboration with Dr. Roger Woodgate of NICHD, we determined the crystal structure of a Y-family DNA polymerase, Dpo4 from S. Solfotaricus, in complex with undamaged DNA and an incoming nucleotide in 2001. These crystal structures provide the first atomic view of the Y-family polymerase in action and reveal a molecular mechanism for the low fidelity DNA synthesis and bypassing modified DNA bases. Recently, we have crystallized Dpo4 in complex with damaged or mispaired DNA. The new structures suggest that Dpo4 binds an incoming nucleotide independent of correct base pairing with the template strand and shuffles the template strand to find a partner for the incoming nucleotide before catalyzing the nucleotidyl-transfer reaction. We propose tht this "free-loading" of nucleotide serves Dpo4 to bypass DNA lesions as well as to make low fidelity DNA synthesis.